You have identified a novel gene (NIMX1) in Xenopuswhich has sequence similarity

Question

You have identified a novel gene (NIMX1) in Xenopuswhich has sequence similarity to members of the FGF family. You have produced recombinant NIMX1 protein and a highly specific antibody that recognises the endogenous protein. Your analysis indicates that NIMX1 is expressed exclusively in the mesoderm of early gastrula stage embryos and NIMX1 expression disappears by the end of gastrulation.

a) How would you experimentally determine whether NIMX1 is a secreted molecule?

b) How would you determine whether, like the FGFs, NIMX signals through the MAP kinase pathway in cells of the early embryo?

c) Briefly describe the experiments and analyses that you would use to determine the subset of genes that require NIMX1 function for their expression in normal development.

Explanation / Answer

a) The most obvious approach from the lectures is to use the oocyte translation system. Inject mRNA coding for NIMX1 into an oocyte and use the antibody to detect if the protein is secreted into the oocyte culture medium. (Other viable approaches will be given appropriate credit.

b) The most obvious approach from experiments discussed in the module would be to treat cells from the embryo with the recombinant protein and carry out a western blot to look for increased levels of diphospho-ERK in the presence of NIMX1.

c) This will require some approach to NIMX1 inhibition. The most obvious approach discussed in the lectures would be the use of antisense morpholino oligos directed against NIMX1 (any other sensible approach will attract credit).
The experimental design would then be to compare gene expression in control versus NIMX1knockdown embryos. Given the known expression profile the best stage to do this would be at early gastrula stages. The most
unbiased approach would be a global transcriptomics analysis, from lectures most likely arrays, but RNA seq would be just as viable. Hopefully some mention will be made of the need to carry out biological replicates to
allow statistical analysis. Some discussion of the criteria that might be adopted in compiling gene list e.g. fold change cutoffs and statistical significance should be included.

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